Adenosine diphosphoribose polymerase binding nitroso aromatic compound useful as retroviral inactivating agents, anti-retroviral agents and anti-tumor agents

ABSTRACT

The subject invention provides for novel compounds for inactivating viruses. These compounds include 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide, 5-nitroso-1(2H)-isoquinolinone, 7-nitroso-1(2H)-isoquinolinone, 8-nitroso-1(2H)-isoquinolinone. The invention also provides for compositions containing one or more of the compounds, and for methods of treating viral infections, cancer, infectious virus concentration with the subject compounds and compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 08/087,566, filed Jul. 2,1993; which is a CIP of application Ser. No. 07/965,541 filed Nov. 2,1992, U.S. Pat. No. 5,516,991, which is a CIP of application Ser. No.07/893,429 filed Jun. 4, 1992; which is a CIP of application Ser. No.07/780,809 filed Oct. 22, 1991, both abandoned.

FIELD OF THE INVENTION

The present invention relates generally to the field of retroviraltherapeutic and inactivating agents and their use in treating retroviralinfections and cancers. More specifically it relates to thosetherapeutic and inactivating C-nitroso compounds which destabilize zincfingers.

BACKGROUND OF THE INVENTION

The enzyme ADP-ribose transferase (ADPRT) (E.C.4.2.30) is achromatin-bound enzyme located in the nucleus of most eukaryotic cells.The enzyme catalyzes the polymerization of the ADP-ribose moiety ofnicotinamide adenine dinucleotide (NAD⁺) to form poly (ADP-ribose). Thepolymer is covalently attached to various nuclear proteins, includingthe polymerase itself.

The many varied roles that ADP-ribosylation plays in cellular metabolismhave made ADPRT a target for drugs essentially useful for combatingneoplasia and viral infections. Numerous physiological activities havebeen detected for compounds that inhibit the polymerase activity ofADPRT. Such activities include a cell cycle dependent prevention ofcarcinogen-induced malignant transformation of human fibroblasts (Kun,E., Kirsten, E., Milo, G. E. Kurian, P. and Kumari, H. L. (1983) Proc.Natl. Acad. Sci. USA 80: 7219-7223), conferring also carcinogenresistance (Milo, G. E., Kurian, P., Kirsten, E. and Kun, E. (1985) FEBSLett. 179: 332-336), inhibition of malignant transformation in hamsterembryo and mouse C3H10T1/2 cell cultures (Borek, C., Morgan, W. F., Ong,A. and Cleaver, J. E. (1984) Proc. Natl. Acad. Sci. USA 81: 243-247),deletion of transfected oncogenes from NIH 3T3 cells (Nakayashu, M.,Shima, H., Aonuma, S., Nakagama, H., Nagao, M. and Sugimara, T. (1988)Proc. Natl. Acad. Sci. USA 85: 9066-9070), suppression of the mitogenicstimulation of tumor promoters (Romano, F., Menapace, L. and Armato, V.(1983) Carcinogenesis 9: 2147-2154), inhibition of illegitimate DNArecombinations (Waldman, B. C. and Waldman, A. (1990) Nucl. Acids Res.18: 5981-5988) and integration (Farzaneh, F., Panayotou, G. N., Bowler,L. D., Hardas, B. D., Broom, T., Walther, C. and Shall, S. (1988) Nucl.Acids Res. 16: 11319-11326), induction of sister chromatid exchange(Ikushima, T. (1990) Chromosoma 99: 360-364) and the loss of certainamplified oncogenes (Grosso, L. E. and Pitot, H. C. (1984) Biochem.Biophys. Res. Commun. 119: 473-480; Shima, H., Nakayasu, M., Aonums, S.,Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci. USA 86:7442-7445).

Compounds known to inhibit ADPRT polymerase activity include benzamide(Kun, E., Kirsten, E., Milo, G. E. Kurian, P. and Kumari, H. L. (1983)Proc. Natl. Acad. Sci. USA 80: 7219-7223), substituted benzamides(Borek, C., Morgan, W. F., Ong, A. and Cleaver, J. E. (1984) Proc. Natl.Acad. Sci. USA 81: 243-247; Romano, F., Menapace, L. and Armato, V.(1983) Carcinogenesis 9; 2147-2154; Farzaneh, F., Panayotou, G. N.,Bowler, L. D., Hardas, B. D., Broom, T., Walther, C. and Shall, S.(1988) Nucl. Acids Res. 16: 11319-11326.; Grosso, L. E. and Pitot, H. C.(1984) Biochem. Biophys. Res. Commun. 119: 473-480; Shima, H., Nakayasu,M., Aonums, S., Sugimura, T. and Nagao, M. (1989) Proc. Natl. Acad. Sci.USA 86: 7442-7445), 3-aminonaphthylhydrazide (Waldman, B. C. andWaldman, A. (1990) Nucl. Acids Res. 18: 5981-5988), isoquinoline,quercetin, and coumarin (1,2-benzopyrone) (Milo, G. E., Kurian, P.,Kirsten, E. and Kun, E. (1985) FEBS Lett. 179: 332-336). Theanti-transforming and anti-neoplastic effect of 1,2 benzopyrone weredemonstrated in vitro and in vivo (Tseng, et al., (1987) Proc. Natl.Acad. Sci. USA 84: 1107-1111).

Other known ADPRT polymerase activity inhibitors include5-iodo-6-amino-1,2-benzopyrone as described in U.S. patent applicationSer. No. 600,593, filed Oct. 19, 1990 entitled "Novel5-Iodo-6-Amino-1,2-Benzopyrones and their Metabolites Useful asCystostatic and Anti-Viral Agents" for use as anti-tumor and anti-viralagents. The cited patent discusses the possibility of using5-iodo-6-nitroso-1,2-benzopyrone as an anti-tumor or anti-viral agent.

The 6-nitroso-benzopyrones have not been hitherto known or described.The only remotely related compounds found in the literature are6-nitro-1,2-benzopyrone and 6-amino-1,2-benzopyrone (6-ABP) (J. Pharm.Soc. Jap., 498: 615 (1923)) for which, only scarce medicinal evaluationhas been reported. In particular, testing was done for sedative andhypnotic effects (J. Pharm. Soc. Japan, 73: 351 (1953); Ibid, 74: 271(1954)), hypothermal action (Yakugaku Zasshi, 78: 491 (1958)), andantipyretic, hypnotic, hypotensive and adrenolytic action (Ibid, 83:1124 (1963)). No significant application for any of these compounds hasbeen described except for 6-ABP.

2-nitrosobenzamide (Irne-Rasa, K. M. and Koubek, E. (1963) J. Org. Chem.28: 3240-3241), and 4-nitrosobenzamide (Wubbels, G. G., Kalhorn, T. F.,Johnson, D. E. and Campbell, D. (1982) J. Org. Chem. 47: 4664-4670),have been reported in the chemical literature, but no commercial use ofthese isomers is known. Neither of these articles suggest the use ofnitrosobenzamides as ADPRT inhibitors.

The anti-retroviral and anti-tumorigenic actions of substituted andunsubstituted 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyroneis the subject of copending U.S. patent applications Ser. No. 585,231filed on Sep. 21, 1990 entitled "6-Amino-1,2-Benzopyrones Useful forTreatment of Vital Diseases" and Ser. No. 600,593 filed on Oct. 19, 1990entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their MetabolitesUseful as Cytostatic and Antiviral Agents", which are incorporatedherein by reference.

The precursor molecule, 1,2-benzopyrone (coumarin), was shown to be aninhibitory ligand of adenosinediphosphoribosyl transferase (ADPRT), aDNA-binding nuclear protein present in all mammalian cells (Tseng, etal., (1987) Proc. Nat. Acad. Sci. USA, 84: 1107-1111).

Hakam, et al., FEBS Lett., 212: 73 (1987) has shown that6-amino-1,2-benzopyrone (6-ABP) binds specifically to ADRPT at the sitethat also binds to DNA, indicating that both 6-ABP and DNA compete forthe same site on ADPRT. Synthetic ligands of ADPRT inhibit DNAproliferation, particularly in tumorigenic cells, (Kirsten, et al.,(1991) Exp. Cell. Res. 193: 1-4). Subsequently, these ligands were foundto inhibit viral replication and are the subject of the copending U.S.patent application entitled "6-Amino-1-2-Benzopyrones useful forTreatment of Vital Diseases," Ser. No. 585,231, filed on Sep. 21, 1990which is hereby incorporated by reference.

Retroviral nucleocapsid (NC) proteins and their respective gagprecursors from all strains of known retroviruses contain at least onecopy of a zinc-binding polypeptide sequence of the type Cys-X₂ -Cys-X₄-His-X₄ -Cys (CCHC) (Henderson, et al., Biol. Chem. 256: 8400-8406(1981)), i.e., a zinc finger domain. This CCHC sequence is essential formaintaining retroviral infectivity (Gorelick, et al., Proc. Natl. Acad.Sci. USA 85: 8420-8424 (1988), Gorelick, et al., J. Virol. 64: 3207-3211(1990)), therefore, it represents an attractive target for retroviralchemotherapy. The HIV-1 gag proteins function by specifically binding tothe HIV-1 RNA, anchoring it to the cell membrane for budding or viralparticles (Meric, et al., J. Virol. 63: 1558-1658 (1989) Gorelick, etal., Proc. Natl. Acad. Sci. USA 85: 8420-8424 (1988), Aldovini, et al.,J. Virol. 64: 1920-1926 (1990), Lever, et al., J. Virol. 63: 4085-4087(1989)). Site-directed mutagenesis studies demonstrated thatmodification of Cys or His residues results in defective viral RNApackaging and noninfectious viral particles are formed (Aldovini, etal., J. Virol. 64: 1920-1926 (1990), Lever, et al., J. Virol. 63:4085-4087 (1989)). The highly abundant nonhistone nuclear protein ofeukaryotes, poly (ADP-ribose) polymerase (E.C.2.4.4.30), also containstwo CCHC-type zinc fingers located in the basic terminal polypeptidedomain, as analyzed by site directed mutagenesis (Gradwohl, et al.,Proc, Natl. Sci. USA 87: 2990-2992 (1990)).

Published experiments have shown that aromatic C-nitroso ligands of poly(ADP-ribose) polymerase preferentially destabilize one of the two zincfingers coincidental with a loss of enzymatic activity but not DNAbinding capacity of the enzyme protein (Buki, et al., FEBS Lett. 290:181-185 (1991)). Based on the similarity to results obtained bysite-directed mutagenesis (Gradwohl, et al., Proc. Natl. Acad. Sci. USA87: 2990-2992 (1990)), it appears that the primary attack of C-nitrosoligands occurred at zinc finger FI (Buki, et al., FEBS Lett. 290:181-185 (1991)). A selective cytocidal action of the C-nitroso groupcontaining poly (ADP-ribose) polymerase ligands on cancer cells wassubsequently discovered (Rice et al., Proc. Natl. Acad. Sci. USA 89:7703-7707.

Based on the coincidental occurrence of the CCHC type zinc fingers inboth retroviral NC proteins and in poly (ADP-ribose) polymerase and theobserved chemotherapeutic effects of C-nitroso-containing ligands oncancer cells, experiments were initiated to test if the C-nitrosocompounds also have antiviral effects on retroviruses containing NCproteins. As described herein experiments testing this hypothesis withthe polypeptide corresponding to the N-terminal CCHC zinc finger ofHIV-1 NC protein, Zn (HIV1-F1) (South, et al., Am. Chem. Soc. 111:395-396 (1989), South, et al., Biochem. Pharm. 40: 123-129 (1990),Summers, et al., Biochemistry 29: 329-340 (1990)), intact HIV-1 virionsand on the propagation of HIV-1 in human lymphocytes in culture, wereperformed.

SUMMARY OF THE INVENTION

The subject invention provides for novel anti-tumor compounds,anti-retroviral compounds and retroviral inactivating compounds. Thesecompounds include 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide,2-nitrosobenzamide, 4-nitrosobenzamide, 5-nitroso-1(2H)-isoquinolinone,7-nitroso-1(2H)-isoquinolinone, 8-nitroso-1(2H)-isoquinolinone.

The invention also provides for compositions containing one or more ofthe compounds, and for methods of treating retroviral infections andcancer with these compounds and compositions.

Also provided for are methods of treating cancer and retroviralinfections with 2-nitrosobenzamide, 3-nitrosobenzamide and4-nitrosobenzamide. Compositions containing one or more of thesecompounds are also provided.

Another aspect of the invention is to provide methods for inactivatingviruses, especially retroviruses, in biological materials, e.g., blood,by adding various nitroso compounds including 6-nitroso-1,2-benzopyrone,2-nitrosobenzamide, 3-nitrosobenzamide, 4-nitrosobenzamide,5-nitroso-1(2H)-isoquinoline, 7-nitroso-1(2H)-isoquinoline and8-nitroso-1(2H)-isoquinoline.

Another aspect of the invention is to provide methods for inactivatingAZT resistant viruses, in particular HIV and SIV by adding variousnitroso compounds including 6-nitroso-1,2-benzopyrone,2-nitrosobenzamide, 3-nitrosobenzamide, 4-nitrosobenzamide,5-nitroso-1(2H)-isoquinoline, 7-nitroso-1(2H)-isoquinoline and8-nitroso-1(2H)-isoquinoline.

Another aspect of the invention is to provide methods for reducing thelevel of integrated viral DNA from the genome of a host, in particularintegrated HIV DNA in a mammalian host, by adding various nitrosocompounds including 6-nitroso-1,2-benzopyrone, 2-nitrosobenzamide,3-nitrosobenzamide, 4-nitrosobenzamide, 5-nitroso-1(2H)-isoquinoline,7-nitroso-1(2H)-isoquinoline and 8-nitroso-1(2H)-isoquinoline.

An additional aspect of the subject invention is to provide novelcompositions of biological materials comprising biological material andthe compounds used in the subject methods.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph comparing the degree of ADRPT polymerase activity(ADPRP) inactivation exhibited by different concentrations of6-nitroso-1,2-benzopyrone, 3-nitroso-benzamide, andnitroso-1(2H)-isoquinolinones (NOQ) (a mixture of the 5 and 7 nitrosoisomers).

FIGS. 2A-2F are a composite of graphs displaying the inhibitory effectsof the ADRPT ligands on (FIG. 2A) 855-2 cells (a cell line of humanB-cell lineage acute lymphoblastic leukemia), (FIG. 2B) H9 cells (a cellline of human T-cell lineage acute lymphoblastic leukemia), (FIG. 2C)HL-60 cells (a cell line of human acute nonlymphoblastic leukemia) and(FIG. 2D) K562 cells (a cell line of human chronic myelogenousleukemia). These cells were cultured while under the influence of thegrowth factors in 10% fetal bovine serum (FCS), whereas in (FIG. 2E) and(FIG. 2F) the 855-2 cells were cultured in the presence of autocrinegrowth factor activity (AGF) or low molecular weight-B-cell growthfactor (BCGF, a T-cell derived lymphokine), respectively.

FIG. 3 is a graph showing the inhibition of increasing levels ofleukemic cell growth (in response to increasing concentrations of FCS)of 855-2 cells by 6-nitroso-1,2-benzopyrone (NOBP) and3-nitrosobenzamide (NOBA).

FIG. 4 is a graph showing that NOBP and NOBA inhibit the ability ofhuman leukemic cells (855-2 and HL-60) to form colonies (CFU) fromsingle cells in a semi-solid medium.

FIGS. 5A-5B show graphs of the relative inhibitory effects ofanti-leukemic doses of ADRPT ligands on the ability of (FIG. 5A) normalrhesus bone marrow stem cells or (FIG. 5B) human peripheral blood stemcells to form colonies in soft agar. Note that the NOBP and NOBA hadminimal effect on normal cells.

FIGS. 6A-6D show graphs displaying the inhibitory effects of NOBP, NOBAand NOQ on four human brain tumor cell lines.

FIG. 7 is a graph comparing the effectiveness of NOBP with vincristine.

FIG. 8 is a graph displaying the effects of NOBP, NOBA and NOQ on humanbreast tumor cell line MDA 468.

FIG. 9 is a graph displaying the effects of NOBP, NOBA and NOQ on murineleukemia cell line L 1210.

FIG. 10 shows the downfield region of the ¹ H NMR spectrum obtained forZn (HIV1-F1)(1 mM in D₂ O solution, pH=6.2, T=30° C.)(bottom) and uponaddition of NOBA (2 mM). The * and + symbols denote the aromatic protonsignals of the zinc-coordinated and zinc-free His 9 residue. Uponcompletion of the reaction, reflected by complete ejection of zinc (t=90min), 50% of the NOBA remained unreacted indicating a 1:1 reactionstoichiometry. NOBA (3-nitrosobenzamide), was synthesized describedelsewhere in this application.

FIG. 11 shows selected regions of the ¹ H NMR spectrum obtained for Zn(HIV1-F1)(bottom) and a synthetic oligonucleotide with sequencecorresponding to a region of the HIV-1 Psi-packaging signal,d(ACGCC)(2nd from bottom). The downfield regions of the spectra show thesignals of the aromatic and ribose H1' protons, and the upfield regioncontain signals of the methyl group protons. Dramatic spectral changesoccur upon addition of oligonucleotide to Zn (HIV1-F1), including largeupfield shifts of the Phe 2 and Ile 10 side chain=n signals and adownfield shifting and broadening of the guanosine-H proton signal(second from the top). After incubation with two equivalents of NOBA,the spectral features are characteristic of metal-free peptide anddissociated nucleic acid (top), indicating that NOBA-induced zincejection leads to loss of high-affinity nucleic acid binding function.

FIG. 12 shows the proposed mechanism for the ejection of Zn⁺² from Zn(HIV1-F1) by NOBA (3-nitrosobenzamide).

FIG. 13 A. shows the HIV-1 inactivation assay using NOBA and NOBP(6-nitroso-1,2-benzopyrone). The HIV-1 stock (HIV-1 100,000 TCID₅₀ wastreated for 30 Min. with 100 μM NOBA or 100 μM NOBP at 22° C., themixture was serial 10-fold diluted and inoculated into PBL cultures.After 9 days the culture supernatants were harvested and the frequencyof infected cultures was measured by immunoassay. The percent positiveof cultures was then plotted as a function of the virus input titer. Therelative amount of infectious virus available to cause 50% infectedcultures was decreased by 4 log units with NOBA. NOBP was synthesized bythe methods described below.

FIG. 13 B. shows an HIV-1 inactivation assay using NOBA at differenttemperatures. The assay was performed as described in FIG. 13A exceptthat the 30 min. preincubation of virus with NOBA was carried out at 0°,22° or 37° C.

FIG. 13 C. shows the dose-responsive effect of NOBA on PHA-PBLviability. PHA-PBL (10⁶ /ml) were treated with increasing doses of NOBAfor 24 hours in the presence of MTT substrate and the relativeabsorbance at 550 nm reflects the metabolic activity of the cells. Thelevel of product formation in the absence of NOBA was considered to be100% and all experimental values were normalized to that control value.

FIGS. 14A-B. The effect of NOBA on SIV_(mac) 239 replication (FIG. 14A)and CEM x174 cell viability (FIG. 14B). Each bar expresses the means of3 independent tests, which do not differ ±10% (not shown). In FIG. 14A,ordinate=p27 antigen assay (ELISA) performed on day 10;abscissa=concentration of NOBA or DMSO. In FIG. 14B, cell viability testdetermined on day 10 by the tetrazolium assay first line bars=virusinfected cells (SIV) in presence of NOBA; second line bars(controls)=uninfected cells treated with NOBA.

FIGS. 15A-B. Analysis of the cellular genome of SIV-infected anduninfected cultures by PCR. CEM x174 cells from 6-day cultures of theexperiment described in FIGS. 14A-B were used for DNA extraction.(Six-day cultures were used instead of 10-day cultures to ensure thepresence of enough extractable DNA). FIG. 15A shows the amplification ofSIV p27 core antigen protein with gag-selective primers. FIG. 15B showsthe amplification of ubiquitous B-actin gene.

FIGS. 16A-B. Effect of NOBA on AZT-resistant strains of SIV. Peripheralblood mononuclear cells (1.2×10⁶) from an SIV_(mac) 239-infected rhesusmacaque (MMU23740) were co-cultivated in a 24-well tissue culture platewith 3×10⁵ CEM x174 cells/ml for 6 days. Alquots (500 ul) of theco-cultivation supernatant were added to 3×10⁵ fresh CEM x174 cells/ml.The cells were incubated at 37° C. for another 2 days (until syncytiaappeared) before adding NOBA or AZT. Cultures were replenished with newmedium containing drug on day 9. Fresh CEM x174 cells (1.75×10⁵ /well)and drug were added on day 13. (FIG. 16A) Virus titers in thesupernatant of cultures 16 days from initial co-cultivation asdetermined by the SIV p27 core antigen capture ELISA. (FIG. 16BB)Viability of cell cultures 16 days from initial co-cultivation asdetermined by the MTT assay. Data presented are the average of duplicatewells.

FIG. 17. The effect of NOBA on SIV assayed in human lymphocytes. Aconcentrated stock of SIV_(sum) (TCID₅₀ =3300) was incubated 30 min at37° C. with 50 μM NOBA. Afterwards, the mixture was serial 10-folddiluted to yield the 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵ dilutions. Eachdilution was used to infect 10⁶ PHA-PBL for 18 hrs at 37° C. after whichthe free virus and drug were removed. Cells were then aliquoted into 96well plates (10⁵ cells/well with 10 replicates per dilution). Cultureswere scored positive for infection if their absorbance at 490 nm in theantigen capture ELISA was >3 S.D. above the mean absorbance 10uninfected cultures. The untreated virus (□) scored positive cultureswith dilutions as low as 10⁻⁴, whereas NOBA-treated virus □ did notscore positive with any cultures.

FIG. 18. Inhibition of HIV-1_(WeJo) replication by NOBA. Human PBMC werealiquoted into 96-well plates (10⁵ /well) and various amounts of NOBAwere added, immediately followed by the addition of 250 TCID₅₀ ofHIV-1_(WeJo), and then cultured for 7 days. Virus production wasmeasured by p24 antigen capture (-▴-) and is expressed as the percent ofantigen in the infected cells in the absence of drug (10 replicates perpoint). Cell viability (-▪-) was determined by the BCECF assay andactivities are expressed as a percentage of the signal in the drug-freeand virus-free control (10 replicates per point).

FIG. 19. HIV-1 Intergrase protein (2 picomoles/reaction) produced via anE. coli. expression vector was stored at -70° C. in 1M NaCl, 20 mM HEPES(pH 7.6), 1 mM EDTA, 1 mM dithiorhreitol, and 20% glycerol (W/V).-⋄-NOBA, preincubation, DNA cleavage assay; -▴- NOBA, preincubation,INTEGRATION assay; -⋄-NOBA, no preincubation, cleavage assay; - - NOBA,no preincubation, INTEGRATION assay; - - Caffeic acid (phenethylester),no pre-incubation, cleavage assay; -▪- Caffeic acid (phenethylester), no-re-incubation, INTEGRATION assay.

FIG. 20. PCR analysis of the effect of NOBA on HIV-1 proviral DNAformation in PBMC. Various concentrations of NOBA were added to aconcentrated stock of HIV-1 (30 min at 37° C.), which was then mixedwith a pellet of 3×10⁶ PBMC and incubated for 24 hours. Theconcentration of drug in the final mixture is indicated. Afterincubation the samples were analyzed by PCR as described in Materialsand Methods. As a control, cells were exposed to virus in the presenceof anti-T4 monoclonal antibodies (T4A) to block infection. The negativecontrol represents the PCR reaction performed in the absence of primers.The positive control is the 8E5 bone marrow isolate from a patientinfected with HIV-1_(LAV).

FIG. 21. Inhibition of endogenous reverse transcription in HIV-1 virionsby NOBA. Permeabilized HIV-1 virions were allowed to reverse transcribetheir native RNA to viral DNA in the absence or presence of variousconcentrations of NOBA. The controls were virus alone (C) or virus inthe presence of 1% DMSO (0 drug). The [³² P]-dCTP labeled transcriptswere viewed by autoradiography on 1% gels.

DESCRIPTION OF SPECIFIC EMBODIMENTS Definitions

The term "biological material" as used herein, refers to any biologicalmaterial extracted from a living organism, including blood, plasma,cerebrospinal fluid, organs, and the like, as well as the processedproducts of biological material extracted from a living organism.

The term "biological composition" as used herein, refers to acomposition comprising a biological material and a compound of interest.

The term "cancer" as used herein, refers to malignant tumors consistingof cells that do not follow normal control signals for proliferation orpositioning.

The term "retrovirus" as used herein refers to RNA viruses which utilizethe enzyme reverse transcriptase to transcribe infecting RNA chains intoDNA complements.

The term "zinc finger" refers to a structural domain of a proteincapable of binding a zinc atom. The nature of zinc finger proteinsdomains is well described in the literature, e.g., Klug and Rhodes,Trends in Biochemical Sciences 12: 464-469 (1987).

The Invention

The subject invention provides for several nitroso compounds that areADPRT polymerase activity inhibitors. These compounds find use asanti-tumor and anti-viral compounds.

Compound (I) has the following formula: ##STR1## wherein R₁, R₂, R₃, R₄,R₅, and R₆ are selected from the group consisting of hydrogen andnitroso, and only one of R₁, R₂, R₃, R₄, R₅, and R₆ is a nitroso group.

A preferred embodiment of compound I is where R₄ is the nitroso group,i.e., the molecule 6-nitroso-1,2-benzopyrone.

Compound II has the formula: ##STR2## wherein R₁, R₂, and R₃ areselected from the group consisting of hydrogen and nitroso, and only oneof R₁, R₂, and R₃ is a nitroso group.

Compound III has the formula: ##STR3## wherein R₁, R₂, R₃, R₄, and R₅are selected from the group consisting of hydrogen and nitroso, and onlyone of R₁, R₂, R₃, R₄, and R₅ is a nitroso group.

Preferred embodiments of compound III are where either R₂ or R₄ is thenitroso group, i.e., 7-nitroso-1(2H)-isoquinolinone and5-nitroso-1(2H)-isoquinolinone, respectively.

The disclosed synthesis for 5-nitroso-1(2H)-isoquinolinone may produce 2closely related structural isomers, 7-nitroso-1(2H)-isoquinolinone and8-nitroso-1(2H)-isoquinolinone. Although experiments testing thebiological activity of 5-nitroso-1(2H)-isoquinolinone may have containedsignificant quantities of 8-nitroso-1(2H)-isoquinolinone or7-nitroso-1(2H)-isoquinolinone, all three isomers are believed topossess similar anti-tumor and anti-viral activity on the basis of theirclose structural similarity. This hypothesis may be conveniently testedby separating the isomers by thin layer chromatography or similarmethods, and comparing the anti-tumor and anti-viral activities of theseparated compounds.

Detailed synthesis of 6-nitroso-1,2-benzopyrone, 3-nitroso-benzamide,5-nitroso-1(2H)-isoquinolinone, 7-nitroso-1(2H)-isoquinolinone, and8-nitroso-1(2H)-isoquinolinone, is provided in the example sectionbelow.

In general, the nitroso compounds of the subject of invention may besynthesized by oxidizing a corresponding amino compound to a compound ofthe subject invention by oxidation with 3-chloroperoxybenzoic acid (orother peroxyacids) in ethyl acetate, a halocarbon solvent or in arelatively concentrated solution in dimethyl formamide.

Detailed synthesis of 3-nitrosobenzamide is described in the examplesection below. Synthesis of the other precursor amino compounds aredescribed in the chemical literature. Some of the compounds arecommercially available. Some precursor amino compounds for oxidation tonitroso compounds of the subject invention are as follows:3-amino-1,2-benzopyrone (Spectrum Chemical Mfg. Corp., Gardena, Calif.90248); 4-amino-1,2-benzopyrone (Aldrich, Rare Chemical Catalog);5-amino-1,2-benzopyrone (by reduction of 5-nitro-1,2-benzopyrone, Chem.Abst. 57 16536d (1962)); 7-amino-1,2-benzopyrone (Gottlieb, et al., J.Chem. Soc. Perkin. Trans. II 435 (1979)); 8-amino-1,2-benzopyrone (byreduction of 8-amino-1,2-benzopyrone, Abdel-Megid, et al., Egypt J.Chem. 20: 453-462 (1977)), and 4-amino-1(2H)-isoquinolinone, byreduction of the corresponding 4-nitro analog (Horning, et al., (1971)Can. J. Chem. 49: 2785-2796).

In addition to compounds (I) to (III), the subject inventioncontemplates various structurally related compounds that have similarcarcinostatic and/or anti-viral activities. These structurally relatedcompounds could be conveniently screened on the basis of their highlypotent inhibitory effect on ADPRT polymerase activity. Structurallyrelated compounds of interest include derivatives substituted byadditional nitroso groups and small, e.g., C₁ -C₃ alkyl groups. Also ofinterest are various nitroso substituted structurally relatedheterocyclic rings such as 3,4-dihydro-1(2H)-isoquinolinones,nicotinamides, pthalhydrazides, and 1,3-benzoxazine-2,4-diones.

Another aspect of the compounds of the subject invention are the easewith which they permeate cell membranes and their relative absence ofnon-specific binding to proteins and nucleic acid.

In practice, the ADPRT polymerase inhibitors of this invention, namelycompounds (I) to (III), and any of their pharmaceutically acceptablesalts, may be administered in amounts, either alone or in combinationwith each other, and in the pharmaceutical form which will be sufficientand effective to inhibit neoplastic growth or viral replication orprevent the development of the cancerous growth or viral infection inthe mammalian host.

Administration of the active compounds and salts described herein can bevia any of the accepted modes of administration for therapeutic agents.These methods include systemic or local administration such as oral,parenteral, transdermal, subcutaneous, or topical administration modes.The preferred method of administration of these drugs is intravenous,except in those cases where the subject has topical tumors or lesions,where the topical administration may be proper. In other instances, itmay be necessary to administer the composition in other parenteral oreven oral forms.

Depending on the intended mode, the compositions may be in the solid,semi-solid or liquid dosage form, such as, for example, injectables,tablets, suppositories, pills, time-release capsules, powders, liquids,suspensions, or the like, preferably in unit dosages. The compositionswill include an effective amount of at least one of compounds (I) to(III), or pharmaceutically acceptable salts thereof, and in addition itmay include any conventional pharmaceutical excipients and othermedicinal or pharmaceutical drugs or agents, carriers, adjuvants,diluents, etc., as customary in the pharmaceutical sciences.

For solid compositions, in addition to the compounds (I) to (III), suchexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like may be used. Thecompounds of the subject invention may be also formulated assuppositories using, for example, polyalkylene glycols, for example,propylene glycol, as the carrier.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc., at least one of activecompounds (I) to (III) in a pharmaceutical solution such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, DMSO andthe like, to thereby form the injectable solution or suspension.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and other substances suchas, for example, sodium acetate, triethanolamine oleate, etc.

If desired, the pharmaceutical composition to be administered maycontain liposomal formulations comprising a phospholipid, a negativelycharged phospholipid and a compound selected from chloresterol, a fattyacid ester of chloresterol or an unsaturated fatty acid. Compounds I, IIor III may be encapsulated or partitioned in a bilayer of liposomes ofthe liposomal formulation according to U.S. patent application Ser. No.08/020,035 entitled "Liposomal Formulations and Methods of Making andUsing Same" filed on Feb. 19, 1993 which is incorporated herein byreference.

Parenteral injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions or solid forms suitable for dissolving in liquid prior toinjection.

A more recently devised approach for parenteral administration employsthe implantation of a slow-release or sustained-release systems, whichassures that a constant level of dosage is maintained, according to U.S.Pat. No. 3,710,795, which is incorporated herein by reference.

Any of the above pharmaceutical compositions may contain 0.01-99%,preferably 1-70% of the active ingredient.

Actual methods of preparing such dosage forms are known, or will beapparent to those skilled in this art, and are described in detail inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 17th Edition, 1985. The composition or formulation to beadministered will, in any event, contain such quantity of the activecompound(s) that will assure that a therapeutically effective amountwill be delivered to a patient. A therapeutically effective amount meansan amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated.

The amount of active compound administered will, of course, be dependenton the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration and the judgment of theprescribing physician. However, an effective dosage may be in the rangeof 1 to 12 mg/kg/day, preferably 1 to 5 mg/kg/day, given only for 1-2days at one treatment cycle. Generally, the upper limit for the drugdose determination is its efficacy balanced with its possible toxicity.

The subject invention provides for methods of reducing the titer ofinfectious retroviruses, particularly retroviruses (including theretrovirus HIV-1) in biological materials by inactivating the viruses.Viruses may be inactivated by contact between the compound of interestand the virus. The term "reducing" includes the complete elimination ofall the infectious viruses of interest, as well as a diminution in thetiter of the infectious viruses. It is of particular interest to reducethe number of infectious viruses in biological material that is to beintroduced into a living organism so as to reduce the possibility forinfection. It is also of interest to reduce the titer infectious virusesthat might be present in or on non-biological materials that come intocontact with living organisms, such non-biological materials includesurgical instruments, dental instruments, hypodermic needles, publicsanitary facilities, and the like.

A preferred embodiment of the subject invention is the reduction ininfectious virus concentration in blood.

Another preferred embodiment of the subject invention is theinactivation of AZT resistant viruses and retroviruses.

Yet another aspect of the subject invention is the removal of integratedretroviral DNA from the genome of a host.

Although the effective amount of the viral-inactivating compound used inthe subject method will vary in accordance with the nature of thecompound and the particular material, biological or otherwise ofinterest, a preferred concentration of 3-nitrosobenzamide is about 15micromolar. An effective amount may readily be determined by testing theeffect of a range of concentrations of the compound of interest on theviral titer of a composition containing a virus of interest.

The subject methods of reducing infectious virus concentration inbiological materials inactivate viruses by employing the step of addingan effective amount of compounds I, II or III, or combinations thereof.These nitroso compounds can destabilize Zn⁺² fingers, i.e. eject Zn⁺² ofthe nucleocapsid proteins of viruses, particularly retroviruses.Preferred embodiments of compounds I, II and III for use in inactivatingviruses are 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide,5-nitroso-1(2H)-isoquinoline, 7-nitroso-1(2H)-isoquinoline,8-nitroso-1(2H)-isoquinoline, and 3-nitrosobenzamide. The use of3-nitroso benzamide is particularly preferred.

Another aspect of the invention is to provide for novel compositionsconsisting of biological materials containing an effective amount ofnitroso compounds I, II and III, or combinations thereof. Preferredembodiments of compounds I, II and III for use in the subjectcomposition are 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide,5-nitroso-1(2H)-isoquinoline, 7-nitroso-1(2H)-isoquinoline,8-nitroso-1(2H)-isoquinoline, 2-nitrosobenzamide, 4-nitrosobenzamide,and 3-nitrosobenzamide. The use of 3-nitrosobenzamide is particularlypreferred. The subject biological compositions may have diminished viralconcentrations and may thus be administered with less risk of infectionthan comparable biological materials.

The subject invention also provides for methods of detecting compoundsthat can inactivate viruses, particular retroviruses, by testing for theeffect of Zn⁺² finger destabilizing, i.e. Zn⁺² ejecting, compounds onthe titer of a virus. Virus titer may be measured by well known methodssuitable for measuring the titer of a virus of interest. The ejection ofZn⁺² from a zinc finger domain may be measured, among other methods, byNMR and/or ⁶⁵ Zn⁺² as described herein and as described in Buki, et al.,FEBS. Lett., 290: 181-185 (1991).

The invention having been described, the following examples are offeredto illustrate the subject invention by way of illustration, not by wayof limitation.

EXAMPLES I. Synthesis and Characterization of 6-Nitroso-1,2-Benzopyrone

An example of a method for the preparation of 6-nitroso-1,2-benzopyronesis provided as follows:

To a stirred solution of 6-amino-1,2-benzopyrone hydrochloride (4.00 g,20 mmol) in water (40 ml) at 22° C. was added a solution of sodiumtungstate (5.93 g, 20 mmol) in water (20 ml) followed by 30% aqueoushydrogen peroxide (5 ml) and stirring was continued for 1.5 hours. Theoxidation product was extracted from the green-colored mixture with two100 ml volumes of ethyl acetate, the combined extracts washed with 0.1NHCl (50 ml) and then water (100 ml). The ethyl acetate was removed byrotary evaporation and the residue recrystallized from warm ethanol (250ml).

Analysis of Reaction Product

The green crystals obtained from the recrystallization step (1.48 g, 42%yield) displayed light absorption at 750 nm characteristic of monomericarylnitroso compounds. Mass spectrum: m/z (relative intensity): 175 (M⁺,100), 161 (16.88), 145 (33.77), 133 (10.38), 117 (56.09), 89 (79.71), 63(57.13). High resolution data for the M⁺ peak: calculated for C₉ H₅ NO₃: 175.0268; found: 175.0271 (deviation-1.1 ppm). ¹ H-NMR (CDCl₃, 300MHz) δ (ppm) from TMS: doublet (6.572 and 6.604) H-4 split by H-3;doublet (7.472 and 7.501) H-8 split by H-7; doublet of doublets(7.860/7.866 and 7.889/7.798) H-7 split by H-8 and finely split by H-5;doublet (7.910 and 7.942) H-3 split by H-4; doublet (8.308 and 8.315)H-5 finely split by H-7. UV/VIS spectrum in ethanol, λ max (ε): 750 nm(46), 316 nm (8.96×10³), 274 nm (2.24×10⁴) Melting Point: The compoundpolymerizes above 160° C., blackens and melts in the range of 325°-340°C.

This nitroso-compound may also be prepared by reacting6-amino-1,2-benzopyrone (as the free base) with 3-chloroperoxybenzoicacid in ethyl acetate or halocarbon solvents.

II. Synthesis of 3-nitrosobenzamide

To a stirred solution of 3-aminobenzamide (Aldrich Chemical Co.) (0.476g, 3.50 mmol) in ethyl acetate (50 ml) at ambient temperature was added1.208 g of 3-chloroperoxybenzoic acid (commercial grade, 50-60% purity,Aldrich), whereupon the solution turned green. After 10 minutes themixture was extracted with 0.14M aqueous sodium bicarbonate (58 ml),washed with three successive 40-ml portions of water, dried over sodiumsulfate, then reduced in volume to 20 mL by rotary evaporation andplaced in the freezer (-20° C.), whereupon the product slowly depositedas a light yellow solid during a period of 72 hours (0.180 g, 34%yield).

The 2-nitrosobenzamide and 4-nitrosobenzamide isomers may be similarlyprepared by oxididizing 2-aminobenzamide and 4-aminobenzamide,respectively.

Analysis of Reaction Product

Melting point: The substance darkens above 135° C., softens andapparently polymerizes in the range 150°-160° C., and melts at 240°-250°C. (with decomposition). In solution the compound is green-blue. Massspectrum: m/z (relative intensity): 150 (M⁺, 100), 136 (10.9), 120(77.2), 103 (31.6), 92 (46.5), 85 (22.8), 71 (33.3). High resolutiondata for the M⁺ peak: calculated for C₇ H₆ N₂ O₂ : 150.042928; found:150.042900 (deviation-0.2 ppm). NMR spectrum: ¹ H-NMR (DMSO-d₆, 300 MHz)δ (ppm) from TMS: broad singlet (7.737) N--H; t (7.824, 7.850, 7.875)H-5 split by H-4 and H-6; d (8.059 and 8.086) H-6 split by H-5; d (8.357and 8.383) H-4 split by H-5; s (8.472) H-2. The singlet at 7.737corresponds to 1 proton; the second N--H proton, spectrallynon-equivalent in this compound, is overlaid by the doublet of H-4. Thisdoublet integrates to 2 protons and can be resolved by addition of D₂ Oto the DMSO solution. UV-VIS absorption spectrum in absolute ethanol,λmax (ε): 750 nm (37.6), 304 nm (5.35×10³) and 218 nm (1.50×10⁴). Anabsorption maximum at 750 nm is characteristic of monomeric arylnitrosocompounds.

In another embodiment, 3-nitrosobenzamide is synthesized by dissolving3-aminobenzamide (5.0 g) in N,N-dimethylformamide (DMF) solvent (25 ml)and then chilled in an ice bath. 3-Chloroperoxybenzoic acid (2.1equivalents) is also dissolved in DMF solvent (25 ml) in a 250-ml flaskequipped with a stirrer and thermometer and, as needed, an ice bath.This solution is chilled to 0°-5° C., the ice bath is removed, and toit, with stirring, is added all at once the chilled 3-aminobenzamidesolution. The mixture immediately becomes a transparent brown color, butwithin 0.5 minute turns to a deep green, and within 1.0 minute thetemperature rises to 70° C., at which time the ice bath is reapplied tothe reaction flask whereupon the temperature begins to fall, and isallowed to fall to 25° C., and stirring is continued for a total of 5minutes. Some precipitation occurs (3,3'-azoxybenzamide side-product),thereafter the mixture is chilled to 5° C. for 10 minutes. The chilledmixture is filtered (suction) to remove the azoxy precipitate, and thegreen filtrate is poured into chilled (5°-10° C.) and stirred aqueous0.40M Na₂ CO₃ (200 ml), resulting in a light green suspension, and thesuspension is stirred for an additional 10 minutes at 5°-10° C. toassure maximal product precipitation. Note that the pH of the suspensionis about 8.5, which assures that 3-chlorobenzoic acid is retained in theaqueous solution as the sodium salt. The precipitate is then collectedon a suction funnel and rinsed with deionized water (100 ml). Thismaterial, which is 3-NOBA (mostly as the tan dimer) containing residual3,3'-azoxybenzamide side-product impurity, is then transferred, whiledamp, to a suitable flask and to it is added 50% aqueous acetic acid(200 ml). The mixture is warmed to 65°-70° C. to dissolve the dimer intothe soluble monomeric 3-NOBA (green) and stirred for 5 minutes at 65° C.The azoxy impurity (yellow) is poorly soluble and remains undissolved.The warm mixture is filtered (gravity) to give a clear green filtrate,which is allowed to cool. It is then chilled and placed in therefrigerator freezer (-20° C.) overnight to allow the 3-NOBA toredeposit as the light tan solid dimer. On the following day the solidproduct is collected on a suction filter, rinsed with fresh solvent, andthe product cake is then dried by vacuum under mild warming for severalhours. One typically obtains 2.24 g of dry 3-NOBA containing a trace ofthe azoxy impurity. The product is recrystallized by dissolving it againin 50% aqueous acetic acid (120 ml) and allowing to redeposit overnightin the freezer. After collection, rinsing and drying in vacuo, theweight is 2.08 g. (37% overall yield). TLC shows the material is 3-NOBAwith a trace of the azoxy impurity.

III. Synthesis of Nitroso-1(2M) -isoquinolinones (a mixture of 5-nitrosoand 7-nitroso-isomers)

1(2H)-Isoquinolinone (isocarbostyril) (Aldrich) was nitrated using ageneral method for isoquinoline compounds (C. G. LeFevre and R. J. W.LeFevre, J. Chem. Soc. 1470 (1935)). The nitration product (a mixture ofthe 5-nitro and 7-nitro isomers, as assigned by Y. Kawazoe and Y.Yoshioka, Chem. Pharm. Bull. (Tokyo) 16: 715-720 (1968), although one ofthe isomers could be the 8-nitro isomer) was then reduced to thecorresponding amino-1(2H)-isoquinolinones using a combination ofpotassium borohydride and palladium-on-carbon catalyst in aqueousmethanol. To the resultant amino-1(2H)-isoquinolinones (as free bases)(0.560 g, 3.50 mmol) in ethyl acetate (175 mL) at 30° C. was added 1.208g of 3-chloroperoxybenzoic acid (Aldrich). The mixture became cloudy andafter 20 minutes it was filtered, extracted with 0.14M sodiumbicarbonate (58 mL), washed with two 50-mL portions of water, and driedover sodium sulfate. The volume of the solution was reduced to 50 mL byrotary evaporation and then placed in the freezer (-20° C.), whereuponan orange solid product was deposited (0.102 g).

Analysis of Reaction Product

Melting point: substance darkens above 175° C., softens, blackens andapparently polymerizes above 195° C., and finally melts in the range310°-335° C. NMR analysis: ¹ H-NMR (DMSO-d₆ /D₂ O, 300 MHz) δ (ppm) fromTMS: m (6.723, 6.741, 6.752); m (7.511, 7.518, 7.533, 7.539, 7.547,7.559, 7.577, 7.585); m (7.663, 7.674, 7.686. 7.698, 7.707); d (7.818,7.846). In the absence of D₂ O, the compound also displays a broadsinglet at 11.90 ppm. The isomeric components were analytically resolvedby thin-layer chromatography (silica gel plates, ethyl acetate solvent),giving two bands, R_(f) 0.82 and R_(f) 0.72. Mass spectrum for R_(f)0.82: m/z (relative intensity): 174 (M⁺, 100), 160 (26.8), 144 (93.0),117 (90.8), 97 (21.9), 89 (96.1), 71 (24.1). High resolution data forthe M⁺ peak: calculated for C₉ H₆ N₂ O₂ : 174.042928; found: 174.043200(deviation=-0.3 ppm). For the component having R_(f) 0.72, M⁺,calculated for C₉ H₆ N₂ O₂ : 174.042928; Found: 174.043200(deviation=-1.6 ppm). These data confirm that the compounds aremono-nitroso isomers.

IV. ADPRT Inactivation Studies

The compounds of the subject invention were tested for their ability toinactivate the polymerase activity of adenosinediphosphoribosyltransferase (ADPRT). Assays were performed according to the method ofBuki and Kun, Biochem. 27: 5990-5995 (1988), using calf thymus ADPRT.The assay results as given in Table I provide the I₅₀ (the concentrationof the compound that inhibits enzyme activity 50%) values for ADPRT ofthe nitroso precursor (6-amino-1,2-benzopyrone) and the more potent5-iodo-derivative (Table I, compounds 1 and 2, respectively). Thenitroso compounds (3,4,5 in Table I) are all highly active as anti-tumorand anti-HIV molecules (as shown in later sections) and are effectiveeven after exposure of cells for a period as short as 30 minutes.5-I-6-nitroso-1,2-benzopyrone (compound 6) in these studies has beenshown to be a relatively poor inhibitor of ADRPT (it is believed thatthe iodo substitution deactivates the NO group as an electrophile) andits biological action is 10 times weaker than that of6-NO-1,2-benzopyrone. For these reasons, the compositions of the presentinvention are believed to be superior to 5-I-6-nitroso-1,2 benzopyrone,which has been shown to be a poor permeant molecule.

                  TABLE I    ______________________________________    I.sub.50 data for aromatic inhibitors of ADPRT    No.       Inhibitor          I.sub.50, μM    ______________________________________    1         6-NH.sub.2 -1,2-benzopyrone*                                 370    2         5-I-6-NH.sub.2 -1,2-benzopyrone*                                 41    3         3-NO-benzamide     15    4         5(7)-nitroso-(2H)-isoquinolinone**                                 13    5         6-NO-1,2-benzopyrone                                 40    6         5-I-6-NO-1,2-benzopyrone                                 400    ______________________________________     *biochemical precursor of nitroso compounds 5 and 6     **a mixture of the 5 and 7nitroso compounds     Assay conditions: ADPRT, 0.4 μg; coDNA, 4 μg; inhibitor diluted     between 0.8 and 600 μM, in 50 μl of 50 mM TrisHCl, 50 mM KCl, 5 mM     2mercaptoethanol, 0.5 mM EDTA, 0.1 mM NAD ([32Plabelled), pH 7.5.     Polymerization at 25° C. for 4 minutes.

FIG. 1 illustrates the % inactivation of ADPRT polymerase activityobserved after 2 hours of incubation with the nitroso-compoundinhibitors at several concentrations.

Additional experiments involving the equilibration between ⁶⁵ Zn⁺² andADPRT-bound Zn⁺² suggest that the ADPRT inhibition activity of thenitroso compounds appears to act by destabilizing the protein throughthe ejecting of Zn⁺². (Buki K. G., Bauer P. T., Mendeleyev, F.; Hakam,H. and Kun E. (1991) FEBS Lett. 290: 181-185). The above mechanism ofaction for ADPRT inhibitors is speculative and does not constitute anylimitation on claimed subject matter.

V. Biological Anti-Cancer Activities of Nitrosobenzopyrones,Nitrosobenzamides and Nitroso-isoquinolinones

Experiments were performed in which various human leukemia cell lineswere exposed to increasing concentrations of 6-amino-1,2-benzopyrone(ABP), 5-iodo-6-amino-1,2-benzopyrone (IABP), 6-nitro-1,2-benzopyrone(NO₂ BP), 6-nitroso-1,2-benzopyrone (NOBP), 3-nitrosobenzamide (NOBA) or5(7)-nitroso-1(2H)-isoquinolinone (NOQ) (a mixture of the 5-nitroso and7-nitroso isomers), and the level of [³ H] thymidine uptake wasdetermined as a measure of cellular proliferation. As shown in FIG. 2,for each of the cell lines tested (855-2 cells, FIG. 2A; H9 cells, FIG.2B; HL-60 cells, FIG. 2C; K562 cells, FIG. 2D) the nitroso-containingligands (NOBP, NOBA, NOQ) were able to inhibit ³ H-thymidine uptake inlower molar concentrations than the other compounds. NOBP, NOBA and NOQpowerfully inhibited 3H-thymidine uptake at a concentration of 10 μM, aconcentration at which the other compounds exhibited comparativelyslight inhibitory effects.

Experiments with H9 cells grown in 10% fetal bovine serum (FCS) (FIG.2B) found NOQ to be the most potent inhibitor, demonstrating almostcomplete inhibition at 10 μM levels. MOBP demonstrated about a 30%decrease in thymidine uptake at 10 μM, and an almost complete inhibitionof uptake at 100 μM. NOBA demonstrated about 75% level of inhibition at10 μM, about 85% inhibition at 100 μM, and almost complete inhibition at250 μM. The remaining amino and nitro compounds were significantly lesspotent and did not display complete inhibition until concentrations of1000 μM were reached.

Experiments with K562 cells grown in 10% fetal bovine serum (FIG. 2D)found NOQ and NOBP to be the most potent inhibitors of cell growth. BothNOQ and NOBP resulted in the almost complete inhibition atconcentrations of 10 μM. NOBP was almost as potent as NOQ and producedabout 90% inhibition at a concentration of 10 μM, and almost completeinhibition at a concentration of 100 μM. The other 3 compounds testedwere significantly less potent.

Experiments with 855-2 cells grown in 10% fetal bovine serum (FIG. 2A)found that NOQ, and NOBP produced almost complete inhibition at aconcentration of 10 μM. At a concentration of 1 μM, NOQ producedsomewhat more inhibition than NOBP, and NOBP produced somewhat moreinhibition than NOBA. Experiments using HL-60 cells (FIG. 2C) providedsimilar conclusions. The other 3 compounds tested were significantlyless potent.

The effect of different growth factors on the growth inhibitory effectsof NOBP was tested. 855-2 cells that were grown in media with (1) 10%fetal bovine serum, (2) autocrine growth factor (AGF) and (3) lowmolecular weight-BCGF (a T cell derived lymphokine) were exposed toincreasing concentrations of the ADRPT ligands. The results are providedin FIG. 2(A, E, F). Cells grown in each of the growth factors were allpotently inhibited by the nitroso-containing compounds, withconcentrations of 5 to 10 μM resulting in 100% inhibition, Thus, NOBP,NOBA and NOQ exert potent inhibitory effects regardless of the source ofgrowth factor activity.

In order to exclude the possibility that NOBP and NOBA manifest theirgrowth inhibitory effects through inactivation of growth factors, theeffects of 10 μM NOBP or NOBA (constant concentration) on 855-2 cells inthe presence of increasing concentrations of fetal bovine serum (FCS)were tested (FCS) contains growth factors for 855-2 cells). The data areprovided in FIG. 3. Growth arrest occurs irrespective of theconcentration of FCS. Thus, the mode of action of NOBP, does not appearto be by antagonism of growth factors but at ADPRT sites related toDNA-replication.

In preliminary experiments the propagation of L1210 murine leukemia wasalso tested in vivo and the results showed that NOBA injectedintraperitoneally at a dose of 2 mg/kg twice a day for 6 consecutivedays, causing no toxic effects, prolonged the life of BDF mice from 10days (untreated) to 35 days (end of observation), thus exerting a highlysignificant in vivo chemotherapeutic response. These results aresomewhat surprising in light of the high levels of ascorbic acid presentin mice. Ascorbic acid, a strong reducing agent, reduces 3-NOBA. In itsreduced form 3-NOBA is not as effective at removing zinc from zincfingers. Hence, one would not expect 3-NOBA to be as an effectivechemotherapeutic agent in the presence of high levels of ascorbic acid.

Tumor cell inhibitory concentrations of NOBP and NOBA were shown not toaffect adversely the viability of normal cells. Experiments wereperformed in which the functions of various cancer cells (855-2 andHL-60 leukemia cells, D32, D37 and CRL 7712 glioblastoma cell lines, 186medulla tumor cell line, L1210 murine leukemia cell line, MDA-468 humanbreast tumor cell line) and normal cells (neutrophil leukocytes and bonemarrow or peripheral blood stream cells) were assessed in the absence orpresence of the compounds. The results are shown in FIGS. 4-9. Together,the data indicate that a concentration of 10 μM of thenitroso-containing ligands effectively suppressed cancer cell growth butdemonstrated only modest effects the functions on normal cells.

VI. Toxicity of NOBP

The cytotoxicity of 0, 2 μM, 4 μM, 8 μM and 10 μM NOBP was measured byexamining the effect of the compound on the colony formation (CFU-GM) ofnormal human stem cells (PBSC). The results of the experiments areprovided in FIG. 5B. Toxicity was not detected, even though levels ofNOBP sufficient to block 855-2 cell proliferation completely weretested.

A similar CFU stem cell toxicity assay was performed in whichcomparisons were made between (ABP) 6-amino-1,2-benzopyrone 1 mM, (IABP)5-I-6-amino-1,2-benzopyrone 250 μM, (NO₂ BP) 6-nitro-1,2-benzopyrone(weakly active) 250 μM, NOBP 10 μM, and NOBA 10 μM. The results of theexperiments are provided in FIG. 5A. Whereas the6-amino-1,2-benzopyrone, 5-I-6-amino-1,2-benzopyrone and the 6-nitroderivative were toxic at the tested given doses, the almost ineffective(against tumor cells) 6-nitro derivative and the highly effective(against tumor cells) NOBP and NOBA were non-toxic.

The effects of 10 μm NOBP and NOBA on superoxide generation by normalhuman peripheral blood neutrophil leukocytes was tested. The results areprovided in Table II. Only minor reductions in superoxide generationwere observed.

                  TABLE II    ______________________________________    Effects of 10 μM NOBP and NOBA on the Generation of    Superoxide by Human Neutrophils                nmol O.sub.2.sup.- /hr/10.sup.5 cells                (mean ± S.D., n = 11)    ______________________________________    10.sup.5 PMN + PMA:                  55.9 ± 7.7    +10 μM NOBP                  34.1 ± 14.1    +10 μM NOBA                  44.4 ± 10.0    ______________________________________

VII. Comparative Efficacy Studies

Vincristine, a highly toxic chemotherapeutic compound, is currently usedin the treatment of leukemia and other malignancies. Studies wereperformed in order to determine the concentration of vincristine thatproduces the same level of growth inhibition as 10 μM NOBP, when assayedon 855-2 leukemia cells grown in vitro. Vincristine was tested in dosesof 0.1, 1, 10 and 100 μM. As shown in FIG. 7. 100 μM of vincristine (ahighly toxic concentration) was required to produce the same level ofinhibition as 10 μM of NOBP, thus NOBP is about 10 times more potentthan an equal concentration of vincristine, and is not toxic to normalcells.

Thus certain aromatic nitroso molecules that are also inhibitors ofADPRT polymerase activity may be useful chemotherapeutic cytostaticagents because of their effectiveness combined with low toxicity.

VIII. Anti-HIV action of NOBP, NOBA and NOO on Stimulated HumanLymphoblasts

The ability of NOBP (6-nitroso-1,2-benzopyrone) and NOBA(3-nitrosobenzamide) to inhibit HIV infections were tested using themethods described in the Journal of Immunological Methods 76: 171-183(1985). Exposure to the two drugs was only for 30 minutes at thecommencement of viral infection, and drugs were never re-added. Theresults given in Table III provide the ID₅₀ of HIV titer 10 days afterinfection of cell cultures with HIV. The data in Table III demonstratethat 10 μM of the nitroso-containing ligands causes a three log decreasein the HIV-1 infectivity titer.

                  TABLE III    ______________________________________    Test Sample  Virus Titer (log ID.sub.50) 10 days    ______________________________________    Virus Alone  5.25    +500 μM ABP                 4.50    +250 μM IABP                 4.66    +250 μM NO.sub.2 BP                 4.93    +10 μM NOBP                 2.01    +10 μM NOBA                 1.05    +10 μM NOQ                 1.73    ______________________________________

IX. Cytocidal Activity of ADRPT Ligands--MTT Assay

Experiments were performed to determine if the inhibition ofproliferation of 855-2 cells seen in culture and in soft agar is due tothe cytostatic or cytocidal effect of the nitroso compounds NOBP, NOBA,and NOQ. Cells at 1×10⁵ /ml (concentration used in bone marrow assay)were treated with NOBP, NOBA and NOQ at 1, 2.5, 5 and 10 μm for 2 hoursthen stimulated with 10% fetal calf serum and incubated for 24 hours.MTT (3-[4,5-Dimethyl-2-yl]-2,5-diphenyltetrazolium bromide) at 1 mg/mlwas then added for 16 hours. The absorbance of the pelleted cell wasthen measured at 550 nm after adding DMSO to solubilze the cells.

Results: With 10 μM NOBP, NOBA and NOQ, complete killing was observed in855-2 cells at 100,000/ml.

X. NMR Studies of Zn⁺² Ejection from Zn (HIV1-F1)

To determine if NOBA is capable of ejecting zinc from retroviral-typezinc fingers, NMR studies were performed on a peptide with amino acidsequence corresponding to the N-terminal CCHC zinc finger of the HIV-1NCprotein, Zn (HIV1-F1), South, et al., J. Am. Chem. Soc. 111: 395-396(1989), South, et al., Biochem Pharm. 40: 123-129 (1990), Summers, etal., Biochemistry 29: 329-340 (1990). NMR spectra of Zn (HIV-F1) wereperformed before and after the addition of NOBA (3-nitrosobenzamide).Previous NMR studies have demonstrated that this peptide binds zincstoichiometrically and with high affinity, South, et al., J. Am. ChemSoc. 111: 395-396 (1989), and three-dimensional structural studies haveshown that the peptide adopts a structure that is essentially identicalto the structure of the corresponding region in the intact NC protein,South, et al., Biochemistry 29: 7786-7789 (1990). NMR spectra of Zn(HIV-F1) were performed before and after the addition of NOBA(3-nitrosobenzamide). The down-field region of the ¹ H NMR spectrumshowing the signals due to the aromatic proton of His 9 and Phe 2 isillustrated in FIG. 10 (bottom). Addition of two molar equivalents ofNOBA results in the loss of the signals due to zinc-bound histidine(denoted by *) and the appearance of broad signals representative ofzinc-free histidine (denoted by +). Other signals in the spectrum aredue to NOBA protons. After 90 min., no signals attributable to thezinc-bound His could be detected, see FIG. 10. After 90 minutes, thesignals due to unreacted NOBA were of equal intensity compared to thereacted NOBA signals, indicating that NOBA reacts stoichiometricallywith Zn (HIV1-F1); this finding has been confirmed by additional studieswith one equivalent of NOBA. By comparison, a 10-fold molar excess ofEDTA is required to remove zinc from Zn (HIV-1-F1), Summers, et al., J.Cell Biochem 45: 41-48 (1991).

XI. NMR Studies of Zn (HIV1-F1) Nucleic Acid Binding

Zn (HIV1-F1) has been shown to bind to single-stranded nucleic acidswith sequence specificity, and a highly stable complex with a 5-residueoligonucleotide, d(CACGCC), containing the sequence of a portion of theHIV-1 Psi-packaging signal has been prepared for high-resolutionstructural studies. Experiments have been performed indicating that theaddition of NOBA to this protein-oligonucleotide complex results inejection of zinc with concomitant dissociation of the zinc fingernucleic acid complex, (FIG. 11). These data indicate that the CCHC array(Cys-X₂ -Cys-X-His-X₄ -Cys) of the HIV-1NC protein Zn (HIV1-F1) can bespecifically affected by NOBA so that functional binding to nucleic acidsubstrates is abated. The reaction mechanism, schematically illustratedin FIG. 12, is consistent with the result of NMR analysis in FIGS. 10and 11. The reaction mechanism proposed in FIG. 12 is a useful model butis not intended to limit the scope of the claimed invention.

XII. Zinc Loss Resulting from Treatment of HIV-1 Virions with NOBA

Experiments were performed to determine if NOBA is capable of ejectingzinc from intact virions. HIV-1 (MN strain) was produced, purified andconcentrated as described in Bess et al., J. Virol. 66: 840-847 (1992).The concentrated virus was diluted to 60 times that of culture fluid inTNE buffer (0.01M Tris-HCl, 0.1M NaCl, 1 mM EDTA, pH 7.2) and incubatedwith 3000 or 6000 μM NOBA at 37° C. The virus was then pelleted andwashed with TNE buffer to remove weakly bound zinc. The quantity of zincin the resulting viral pellets was determined as described in Bess etal., J. Virol. 66: 840-847 (1992). No significant loss of viral proteinsin the pellet was detected by p24 and gp120 competitionradioimmunoassays and comassie-stained sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

The data in Table IV demonstrate that treatment of concentratedsuspensions of HIV-1 (60× with respect to culture solution) with NOBAresults in losses of 50-83% of the viral zinc and complete loss ofinfectivity. Since edge x-ray absorption fine structure spectroscopy hasshown that the majority of the zinc in intact retroviruses iscoordinated by the CCHC ligands (Summers, et al., Protein Science 1:563-574 (1992) and Chance et al., Proc. Natl. Acad. Sci. 89: (1992) inpress)), the ejection of zinc from virions by NOBA is directlyattributable to a destabilization of the nucleocapsid CCHC zinc fingers.Anti-HIV activity for R--NH₂ type ligands of poly (ADP-ribose)polymerase (Cole et al., Biochem. Biophys. Res. Commun. 180: 504-514(1991)) may now be attributed, in part, to destabilization of retroviralCCHC zinc fingers since R--NH₂ compounds are metabolic precursors ofR--NO type molecules (Buki, et al., FEBS 2Lett., 290: 181-185 (1991)).

                  TABLE IV    ______________________________________    NOBA  Incubation                   Zinc, Control                              Zinc, NOBA-Treated                                          Zinc Loss    (μM).sup.b          time (h) Sample (μg/ml)                              Sample (μg/ml)                                          (%)    ______________________________________    3,000 2        0.21       0.11        52    6,000 4        0.24       0.04        83    ______________________________________     .sup.b Concentrations correspond to molar NOBA:zinc finger ratios of ca.     350:1 (3,000 μM) and 700:1 (6,000 μM)

XIII. In Vivo Testing of NOBP and NOBA

Both NOBP (6-nitroso-1,2-benzopyrone) and NOBA were tested on viralgrowth of HIV-1 (LAV strain) in phytohemagglutinin-stimulated humanperipheral lymphocytes (PBL) as follows. An HIV-1 stock having aninfectivity titer of 100,000 TCID₅₀ was incubated for 30 min. at 22° C.with either of the drugs. Control HIV samples, containing no drugs, wereincubated in the same manner. Following successive serial dilutions of10-fold that resulted in HIV-1 titers as giver in the lower abscissa ofFIG. 13 (A,B), viral growth was initiated by adding the HIV-1 dilutionsto PBL and allowing an incubation period of 9 days. At the end of thisincubation cultures were assayed for productive infection by animmunoassay for HIV-1 antigens and by reverse transcriptase as describedin McDougal, et al., J. Immunol Methods 76: 171-183 (1985). Virus titerswere expressed as percentile values (percent infected cultures) comparedto the controls, containing HIV dilutions which were not preincubatedwith the C-nitroso drugs. In a separate series of experiments, HIV-1dilutions and C-nitroso drugs (see upper abscissa) were not preincubatedbut were added simultaneously to lymphocytes in exactly the sameconcentrations as described previously (i.e., following preincubation)and viral growth monitored 9 days later. As illustrated in FIG. 13,South, et al., J. Am. Chem. Soc. 111: 395-396 (1989), South, et al.,Biochem Pharm. 40: 123-129 (1990), Summers, et al., Biochemistry 29:329-340 (1990), the inhibition of HIV-1 propagation was profound whenC-nitroso drugs were preincubated with HIV-1, whereas only negligibleeffect on HIV-1 growth occurred when both drugs and virus were addedsimultaneously. Between the two C-nitroso drugs, NOBA induced a greaterdepression of HIV-1 propagation.

Experiments were performed in which the incubations with NOBA wascarried out at 0°, 22°, and 37° C. From FIG. 13B it is apparent thatinactivation of HIV-1 by NOBA occurred maximally at 37° C., suggesting aprobable lesser accessibility of the viral zinc finger to the drugs ascompared to the Zn (HIV-1-F1) present in the isolated polypeptide (FIGS.10 and 11). In agreement with the negligible cytotoxic effect ofC-nitroso drugs on non-tumor cells described elsewhere in thisapplication, human lymphocytes tolerated NOBA up to 50 μM without majorchanges in cell metabolism, which was assayed by quantitative dyereduction as described in Mosaran, J. Imm. Methods 65: 55-63 (1983).

The direct action of C-nitroso drugs on a critical molecular structureof the HIV-1 virus itself, the zinc finger of NC protein, distinguishesthese drugs from any presently known chemotherapeutic agents. Metabolicprecursors of C-nitroso drugs, which are R--NH₂ type ligands of poly(ADP-ribose) polymerase, suppress HIV-1 replication of both MT-2 an Aa-2cells, Cole, et al., Biochem. Biophys. Res. Comm. 10: 504-514 (1991).Correlation between the inhibitory binding of these R--NH₂ ligands topoly (ADP-ribose) polymerase and their anti-HIV effectively indicatesthe participation of this nuclear enzyme in the mode of action of thesemolecules as antiviral agents. However the concentration of the R--NH₂drugs required to block HIV-1 replication is about 10³ higher than theeffective antiviral concentration of C-nitroso drugs. Considering therelatively slow rate of the oxidation of R--NH₂ drugs to C-nitrosomolecules (Buki, et al., FEBS Letters 290: 181-185 (1991)) in cells, therelatively high concentrations (millimolar) of R--NH₂ drugs correspondto their role as "pre-drugs" or sources of C-nitroso type moleculeswhich are effective in micromolar concentrations. Therefore a directaction of C-nitroso drugs formed from their precursors is feasible,although it cannot be ruled out at present that these drugs, besidesacting directly on HIV-1 as shown here--may have an additional mode ofaction that could be related to the effects of C-nitroso drugs asapoptosis-inducing agents in cancer cells.

XIV. Inhibition of the Replication of Native and3'-Azido-2',3'-Dideoxythymidine (AZT)-resistant Simian ImmunodeficiencyVirus (SIV) by 3-NOBA

CEM x174 cells are the fusion product of human B cell line 721.174 andhuman T cell line CEM (12). A molecular clone of SIV_(mac) (SIV_(mac)239) was kindly provided by Dr. R. Desrosiers of the New England PrimateResearch Center. AZT (3'-azido-2',3'-dideoxythymadine) was manufacturedby the Burroughs Wellcome Co. The compound 3-nitrosobenzamide (NOBA) wassynthesized as described in example II. RPMI 1640 supplemented withL-glutamine was purchased from Gibco Labs, Inc.

Preincubation with the 3-NOBA

CEM x174 cells were suspended at 4×10⁵ cells/ml and distributed into23-well tissue culture plates. Cultures were treated with variousconcentrations of the test compound (along with DMSO as controls) andincubated at 37° C. for 1 hr in a CO₂ incubator. The cells were infectedwith 5 μl of a stock solution of SIV_(mac) 239 at 300 TCID₅₀ /ml (50%tissue culture infectious dosage per ml cell suspension). Cell viabilitywas determined by the tetrazolium salt (MTT) assay and the cultures weredivided 1:4 every 3-4 days in medium containing the drug. The cultureswere examined periodically by light microscopy for the presence ofsyncytia. The virus titers were determined by analysis of supernatantSIV p27 core antigen protein or reverse transcriptase (RT) levels.

Preincubation with the Virus

CEM x174 were distributed into 24-well tissue culture plates as above.Cells were incubated with virus for 2 days (until syncytia appeared)before treatment with NOBA. Cultures were examined periodically for thepresence of syncytia. Virus titers were determined by SIV p27 or RTassays.

Reverse Transcriptase Assays

To test for reverse transcriptase activity, 10 μl of infected cellsupernatant was added to a reaction mixture containing 50 mM tris-HCL(pH 8.0), 5 mM MgCl₂, 10 mM dithiothreitol (DTT), 20 mM KCl, and 1%Triton X-100 in a total volume of 50 μ. Poly(rA)oligo(dT)₁₂₋₁₈ waspresent at 100 μg/ml and TTP at 2.4 μM. The reaction mixtures wereincubated at 37° C. for 1 hr and the TCA precipitable radioactivity wasfiltered onto nitrocellulose filters which were then washed, dried, andcounted.

Tetrazolium Salt (MTT) Assays

Cell viability was measured by a published procedure. Hansen et al. J.Immunol Methods 119: 203-210 (1989). Briefly, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytltetrazolium bromide) wasdissolved at a concentration of 5 mg/ml in sterile phosphate bufferedsaline (PBS). Twenty μl of MTT solution were added into each microtiterwell containing 100 μl of cell culture. Following 2 hr incubation at 37°C., 100 μl of solubilizing medium (cf. 13) were added. After overnightincubation at 37° C., optical densities were measured at 570 nm using amicrotiter plate reader.

SIV_(mac) p27 core antigen level was determined by an enzyme immunoassayprovided by Coulter Corp. (Hialeah, Fla.). The assay was performedaccording to the manufacturer's specifications.

Polymerase Chain Reaction (PCR) Analysis of Infected Cell Genomes

DNA extracted from drug-treated, SIV-infected CEM x174 cells or fromcontrol cells were screened for the presence of the SIV sequence by apublished method using individually-designed primers corresponding tothe SIV gag gene. Blackbourn et al., J. Virol Methods 37: 109-118(1992). Northern hybridization has shown the primers to be complementaryto the region encoding the major core antigen protein p27.

Assays on human lymphocytes were performed as described in Example XIII.

The effectivity of NOBA in preventing SIV_(mac) 239 replication in CEMx174 cells was determined by preincubating cells in varyingconcentrations of NOBA at 37° C. for 1 hr before infection with thevirus. As illustrated in FIG. 14A, pretreatment of CEM x174 cells with 0to 20 μM NOBA and maintenance of these drug concentrations during theentire experimental period strikingly abolished SIV_(max) 239replication only at 20 μM NOBA, no effect NOBA was detected at lowerdrug concentrations. The exact reasons for the sharp transition betweenineffective (below 15 μM) and fully effective (20 μM) NOBAconcentrations are not known, but it is possible that the quantity ofintracellular NOBA-inactivating systems may explain this phenomenon,which is overcome by higher than 15 μM NOBA. Coincidental with theantiviral action of 20 μM NOBA cell viability was maintained at thelevel of virus-free controls (FIG. 14B). However, when SIV p27 levelswere high the cytocidal action of SIV_(mac) 239 was reflected in asignificantly depressed cell viability, as would be predicted.

Since virus replication as expected to coincide with the appearance ofintegrated viral DNA in the genome of CEM X174 cells, cellular DNA wasassayed by the polymerase chain reaction (PCR) method with the aid ofspecifically designed gag-selective SIV primers. Blackbourn et al., J.Virol Methods 37: 109-118 (1992). Results of the PCR assay are shown inFIGS. 15A-B. Lane 1 in FIG. 15A is a molecular marker (Hind III digestedφX 174 DNA), and Lane 2 is the plasmid DNA encoding the SIV p27 coreantigen protein amplified by gag specific primers. Lane 3 illustratesthe absence of the specific amplified DNA from non-infected controlcells, and Lanes 4-7 show the result of PCR assay in SIV-infected cellsin the absence of NOBA (Lane 4), with 0.1% DMSO (Lane 5) and afterpreincubation and treatment with 10 μM NOBA (Lane 6) and finally with 20μM NOBA (Lane 7), which completely abolished the signal for theinfectious DNA (compare with FIG. 14A). To rule out the possibleartifact that the absence of SIV gag DNA may be due to incomplete DNAextraction technique, we also tested for the ubiquitous β-actin gene asshown in FIG. 15B, where Lane 1 shows a molecular marker (HindIII-digested φX174 DNA), lane 2 is the β-actin segment amplified byβ-actin specific primers, and lanes 3-7 are β-actin primer amplificationof the DNA extracted from a non-infected cell culture (lane 3) andinfected cell cultures treated with 0 μM NOBA (lane 4), 0 μM NOBA with0.1% DMSO (lane 5), 10 μM NOBA (lane 6), and 20 μM NOBA (lane 7). Theseresults confirm that the absence of the SIV genome in infected cellstreated with 20 μM NOBA was not due to the lack of extractable DNA.

In order to identify an AZT-resistant SIV strain, viruses fromSIV-infected rhesus macaques were isolated and tested for theirresistivity toward AZT. The molecular clone SIV_(mac) 239 wasAZT-sensitive but virus isolates from an SIV_(mac) 239-infected rhesusmacaque (MMU 23740) fourteen months post-infection were AZT resistant;AZT only partially inhibited the growth of SIV 23740 compared toSIV_(mac) 239, suggesting that the macaque virus contained a mixture ofthe original infecting virus (SIV_(mac) 239) and other, mutated viruses.A comparison of the number of syncytia formed in AZT-treated wellsrevealed the complete absence of the cytopathic effect of SIV_(mac) 239in contrast to SIV 23740 (Table V).

                  TABLE V    ______________________________________    The effect of AZT on nonresistant and AZT-resistant    SIV.sub.max as assayed by syncytia formation    Virus        AZT concentration (μM)                                Syncytia.sup.b    ______________________________________    SIV.sub.mac 239                  0             ++++                 10             -                 15             -                 20             -                 25             -                 30             -                 35             -                 40             -    SIV 23740     0             ++++    (AZT-resistant)                 10             ++++                 15             ++++                 20             ++++                 25             ++++                 30             ++++                 35             ++++                 40             ++++    ______________________________________     .sup.a CEM x174 cells (1.5 × 10.sup.5 /500 ul) were infected with     equal doses of SIV.sub.mac 239 or virus isolates from an SIV.sub.mac     239infected rhesus macaque (MMU 23740). Three days postinfection, AZT     concentrations ranging from 0 to 40 μM were added to the cells and the     cultures were incubated for four days. The wells were replenished with     fresh CEM x174 cells and AZT and incubated for an additional three days.     Cell cultures were then examined for syncytia formation.     .sup.b The number of syncytia in cell cultures was counted in arbitrary     fields under 60× magnification and scored as follows: over 30     (++++), 20-30 (+++), 10-20 (++), 1-9 (+), and 0 (-).

The inhibitory action of NOBA on the replication of AZT-resistant SIVstrains was assayed by incubating supernatants of 6-day-oldco-cultivation systems, consisting of MMU23740 PBMCs and CEM x174, withfresh CME x174 cells. This system simulates conditions that may exist invivo. Assays for the p27 core antigen with ELISA 16 days after theinitial co-cultivation showed a NOBA does-dependent depression of SIV23740 production, whereas no antiviral action of AZT occurred (FIG.16A). There was no significant drug-dependent decrease of cell activitydue to either NOBA or AZT (FIG. 16B).

In contrast to the powerful anti-SIV action of NOBA, no direct effect onreverse transcriptase activity could be ascertained (Table VI)

                  TABLE VI    ______________________________________    .sup.3 H-TPP incorporation by SIV 239-RT in the presence of NOBA.sup.a    Concentration of    NOBA (μM)    cpm (× 10.sup.3)    ______________________________________     0 (no enzyme)   0.4     0              761.4     0 (0.1% DMSO)  743.8     0.8            897.0     20.0           763.8     40.0           748.8     80             764.9    400.0           690.7    800.0           706.6    ______________________________________     .sup.a Reverse transcriptase assays were performed with DMSO controls in     the presence or absence of NOBA.

The direct anti-SIV action of NOBA was also assayed with humanperipheral lymphocytes that were stimulated by phytohemagglutinin(PHA-PBL) as described for HIV in Example XIII. This experimentrepresents a direct comparison between SIV and HIV in the same testsystem. As seen in FIG. 17, preincubation of SIV_(smm) with 50 μM NOBAfor 30 min at 37° C. completely suppressed SIV replication in PHA-PBL.As determined in separate studies, designed to quantitate thedose-responsive effect of NOBA on SIV replication in PBMCs, the EC₅₀value (concentration of drug that suppresses 50% virus replication)varied between 17 and 8 μM NOBA for SIV_(smm) and SIV_(smbi) strains,respectively.

XV. The Site of Antiviral Action of 3-nitrosobenzamide on theInfectivity Process of HIV in Human Lymphocytes Virus ReplicationInhibition Assays

Phytohemagglutinin-stimulated human peripheral blood mononuclear cells(PBMC) were distributed into 96-well plates (10⁵ /well) in the presenceof indicated concentrations of NOBA and 250 TCID₅₀ of the HIV-1_(WeJo)pediatric clinical isolate that has been propagated only in human PBMC.After 7 days, cultures were assayed for p24 antigen content using a p24antigen-capture kit (Coulter Immunology, Hialeah, Fla.). Cell viabilitywas quantitated using biscarboxyethyl-5(6)-carboxyfluoresceinacetoxymethyl ester (BCECF, Molecular Probes, Inc., Eugene, Ore.) aspreviously described. Gulakowski et al., J. Virol Methods 40: 347-356(1991).

Enzyme Assays

The in vivo activity of RT was determined with the Boehringer MannheimELISA kit and 3'-azido-3'-deoxythymidine-5'-triphosphate (AZTTP) wasincluded as a positive control for inhibition of RT. For the endogenousreverse transcription assay, 10 μg of virus HIV-1_(IIIB) (UniversalBiotechnology Inc., Rockville, Md.) were treated with NOBA at indicatedconcentrations for 10 min at 25° C., followed by permeabilization of thevirus with melittin (Sigma Chemical Co., St. Louis, Mo.) and subsequentincubation of the reaction mixture for 6 hrs at 39° C. as previouslydescribed. Yong et al., AIDS 4: 199-206 (1990). Reactions wereterminated with 0.1% SDS/10 mM EDTA, and electrophoresis performed on0.7% agarose gels, the gels dried and exposed to autoradiography. HIV-1protease activity was quantitated by a reverse phase HPLC assay aspreviously described (Wondrak et al., FEBS Lett 280: 347-350 (1991)) andHIV-1 integrase activity was measured as reported (Fesen et al.,P.N.A.S. 90: 2399-2403 (1993)). For comparison, topoisomerase I and IIwere assayed as described (Jaxel et al., J. Biol. Chem. 266: 20418-20423(1991)).

DNA Amplification Procedures

Proviral DNA synthesis was monitored with an undiluted HIV-1_(IIIB)stock that had been premixed with NOBA or the DMSO solvent and to thismixture 3×10⁶ PBMC were added and cultured for 24 hrs. Cells were thenwashed and the DNA extracted and PCR-amplified with LTR/gag primer pairs(M667/M661) and the products analyzed by 2% agarose gels which werevisualized by autoradiography of the dried gels, as previously described(Zack et al., Cell 61: 213-222 (1990)).

Virus Attachment Assays

Binding of HIV-1_(RF) to PBMC was measured by a p24-based assay.Briefly, 5×10⁵ PBMC were incubated with a concentrated stock of virusfor 30 min, the unbound virus washed away, and the cell-associated virussolubilized and analyzed by the p24 antigen-capture assay. The bindingof HIV-1 to PBMCs was blocked in a concentration-dependent manner bydestran sulfate (see Table VII. Cell surface binding of HIV-1_(LAV) toPBMC was also quantitated by flow cytometry using FITC-anti-HIV-1_(LAV)as reported McDougal et al., J. Immunol. 135: 3151-3162 (1985).

The 3-Nitrosobenzamide was synthesized as described in Example II.

Inhibitory Effect of NOBAS on Viral Replication

The p7NC protein (nucleocapsid protein of HIV-1 contains two separatezinc fingers sequences that are required not only for packaging of viralgenomic RNA but also for early events in viral replication, suggestingthat NOBA may induce a specific inhibitory effect in early stages ofviral infection. To define this antiviral effect, studies were designedto measure the concentration-dependent action of the drug on HIV-1replication under conditions in which the target cells (PBMC) weresimultaneously mixed with the HIV-1_(WeJo) pediatric clinical isolateand various concentrations of NOBA. As shown in FIG. 18, NOBA inhibitedp24 viral antigen production with an EC₅₀ (level of drug that inhibitsinfection by 50%) of 1.56 μM and there is a depression of lymphocytes at50 μM NOBA. Since the in vitro culturing of lymphocytes requiresphytohemagglutinin, necessarily introducing some degree ofartificiality, in vitro efficacy of NOBA has to be studied in cell typesthat need no artificial growth stimulants. For these reasons theapparent efficacy of NOBA, estimated to be about 32, in stimulatedlymphocytes may be an underestimation.

Insensitivity of the binding of HIV-1 to cells, and of reversetranscriptase, HIV-1 protease and integrase to NOBA. The influence ofNOBA on the binding of HIV-1 to PBMC and on the in vitro activities ofHIV-1 namely on reverse transcriptase (RT), protease (PR) and integrase(IN) was determined. Pretreatment of virus with 100 μM NOBA had noeffect on the attachment of virus, as quantitated by the association ofp24 with the PBMC (Table VI), whereas 10 μg/ml dextran sulfate producednearly complete inhibition. The lack of an effect on viral attachment byC-nitroso drugs was also confirmed by a flow cytometry method which isbased on the FITC-anti-HIV-1 assay (not shown). Employing an artificialhomopolymer template-primer, (poly(rA).oligo(dT)), there was noinhibitory effect of NOBA on the activity of RT (see Table VI), while3-azido-3'-deoxythymidine-5'-triphosphate (AZTTP) effectively inhibitedRT activity. Likewise, although the A-74704 synthetic PR inhibitor (Chowet al., Nature 361: 560-564 (1993)) depressed PR at a concentration of 1μM, NOBA (100 μM) demonstrated no inhibition of PR activity (Table I).It is of particular interest that NOBA had no effect on IN activity(FIG. 19) even after preincubation. This protein contains a "classical"type of zinc finger sequence (CCHH rather than the retroviral CCHC type)Khan et al., Nucleic Acids Research 19: 851-860 (1991). As a positivecontrol, the inhibitory action of caffeic acid (phenethylester) on IN isalso shown. Since the major DNA binding nuclear enzymes, topoisomerase I& II, contain zinc, the action of NOBA was also tested on these enzymes.At concentrations of NOBA which completely block HIV infectivity or theformation of proviral DNA, no effects on topo I and II could beascertained even after preincubation for one hour (FIG. 19). Thus, NOBAwas without effect on four major targets of HIV-1 attachment, RT, PR andIN) and exhibited specificity towards the retroviral zinc fingerstructure.

NOBA blocks the synthesis of proviral DNA. The formation of proviral DNAwithin PBMC was determined by mixing a concentrated stock suspension ofHIV-1_(LAV) with the drug followed by addition to PBMC cultures. After24 hrs. in culture the cells were analyzed by the PCR methodology withLTR/gag (M667/M661) primer pairs to probe for the presence offull-length or nearly full-length proviral DNA Zack et al., Cell 61:213-222 (1990). The products of reverse transcription, as assayed byPCR, were completely blocked by 10 μM NOBA (FIG. 20). Virus replicationwas also blocked under the same conditions (not shown). There wasinhibition of the reverse transcription process by NOBA when assayed inpermeabilized HIV-1 virions (FIG. 21) composed of the native RNAtemplate, tRNA^(lys),3 primer, RT and NC proteins. This "endogenous"assay contained a 100-fold higher concentrated stock of HIV-1_(IIIB)than the tests illustrated in FIG. 20, therefore higher concentrationsof NOBA were required, since there is a stoichiometry between theconcentration of NOBA and that of retroviral zinc fingers. See ExampleX. Even though NOBA does not directly affect the RT enzyme, it preventsthe formation of mature proviral DNA that is required for integrationinto the cellular genomic DNA.

                  TABLE VI    ______________________________________    Effect of NOBA on Various HIV-1 Functions    Condition    Activity.sup.c               Attachment.sup.a                          RT Activity.sup.b                                     PR    ______________________________________    No Drug    1.01 ± 0.09                          1.002 ± 0.108                                     0.335 ± 0.129    100 μM NOBA               1.17 ± 0.22                          1.109 ± 0.037                                     0.375 ± 0.147    10 μg/ml Dex. Sulf               0.06 ± 0.05    1 μM AZTTP         0.087 ± 0.058    1 μM A-74704                  0.005 ± 0.01    ______________________________________     .sup.a Values for virus attachment (mean ± sd, n = 3 of the absorbance     at 450-650 nm) represent p24 levels, as measured by an antigencapture     assay.     .sup.b HIV1 RT activities as the mean ± sd (n = 3) of the absorbance     (405 nm/490 nm).     .sup.c Values represent the mean ± sd (n = 3) of the change in     absorbance at 206 nm for the cleavage of the HIV1 PR synthetic substrate.

All publications, patents, and patent applications cited above areherein incorporated by reference.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the above-described modes for carrying out theinvention which are obvious to those skilled in the field ofpharmaceutical formulation or related fields are intended to be withinthe scope of the following claims.

What is claimed is:
 1. A method of inactivating a virus having a Zn⁺²finger nucleocapsid protein, said method comprising administering aneffective amount of a Zn⁺² finger destabilizing compound wherein saidcompound is selected from the group consisting ofa compound having theformula: ##STR4## wherein R₁, R₂, R₃, R₄, R₅ and R₆ are selected fromthe group consisting of hydrogen and nitroso, and only one of R₁, R₂,R₃, R₄, R₅ and R₆ is a nitroso group, a compound having the formula:##STR5## wherein R₁, R₂, and R₃ are selected from the group consistingof hydrogen and nitroso, and only one of R₁, R₂, and R₃ is a nitrosogroup, and a a compound having the formula: ##STR6## wherein R₁, R₂, R₃,R₄, and R₅ are selected from the group consisting of hydrogen andnitroso, and only one of R₁, R₂, R₃, R₄, and R₅ is a nitroso group.
 2. Amethod according to claim 1, wherein said compound is selected from thegroup consisting of 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide,5-nitroso-1(2H)-isoquinolinone, 7-nitroso-1(2H)-isoquinolinone, and8-nitroso-1(2H)-isoquinolinone.
 3. A method according to claim 2,wherein said compound is 3-nitrosobenzamide.
 4. A method according toclaim 1 wherein said virus is a retrovirus.
 5. A method according toclaim 4 wherein said retrovirus is HIV-1.
 6. A composition comprising abiological material and a compound that destabilizes a Zn⁺² finger on aviral nucleocapsid protein wherein said biological material is blood andwherein said compound is selected from the group consisting ofa compoundhaving the formula: ##STR7## wherein R₁, R₂, R₃, R₄, R₅ and R₆ areselected from the group consisting of hydrogen and nitroso, and only oneof R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group, a compound having theformula: ##STR8## wherein R₁, R₂, and R₃ are selected from the groupconsisting of hydrogen and nitroso, and only one of R₁, R₂, and R₃ is anitroso group, and a a compound having the formula: ##STR9## wherein R₁,R₂, R₃, R₄, and R₅ are selected from the group consisting of hydrogenand nitroso, and only one of R₁, R₂, R₃, R₄, and R₅ is a nitroso group.7. A composition according to claim 6, wherein said compound is3-nitrosobenzamide.
 8. A method for treating retroviral infections, saidmethod comprising the step of administering an effective amount of acompound selected from the group consisting of:a compound having theformula: ##STR10## wherein R₁, R₂, R₃, R₄, R₅ and R₆ are selected fromthe group consisting of hydrogen and nitroso, and only one of R₁, R₂,R₃, R₄, R₅ and R₆ is a nitroso group, a compound having the formula:##STR11## wherein R₁, R₂, R₃, R₄, and R₅ are selected from the groupconsisting of hydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, andR₅ is a nitroso group, and a compound having the formula: ##STR12##wherein R₁, R₂, and R₃ are selected from the group consisting ofhydrogen and nitroso, and only one of R₁, R₂, and R₃ is a nitroso group.9. A method according to claim 8, wherein said compound is selected fromthe group consisting of 6-nitroso-1,2-benzopyrone, 3-nitrosobenzamide,5-nitroso-1(2H) -isoquinolinone, 7-nitroso-1(2H)-isoquinolinone, and8-nitroso-1(2H) -isoquinolinone.
 10. A method according to claim 8,wherein said compound is 3-nitrosobenzamide.
 11. A method according toclaim 8 wherein said retroviral infection is an HIV infection.
 12. Acomposition for the treatment of retroviral diseases, said compositioncomprising a compound according to claim
 8. 13. A pharmaceuticalcomposition for the treatment of retroviral diseases, said compositioncomprising a pharmaceutically effective amount of a compound accordingto claim
 8. 14. A composition for the treatment of HIV infections, saidcomposition comprising a compound according to claim
 8. 15. A method ofinactivating an AZT resistant virus, said method comprisingadministering a pharmaceutically effective amount of a nitroso compoundwherein said compound is selected from the group consisting ofa compoundhaving the formula: ##STR13## wherein R₁, R₂, R₃, R₄, R₅ and R₆ areselected from the group consisting of hydrogen and nitroso, and only oneof R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group, a compound having theformula: ##STR14## wherein R₁, R₂, and R₃ are selected from the groupconsisting of hydrogen and nitroso, and only one of R₁, R₂, and R₃ is anitroso group, and a a compound having the formula: ##STR15## whereinR₁, R₂, R₃, R₄, and R₅ are selected from the group consisting ofhydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, and R₅ is anitroso group.
 16. A method according to claim 15, wherein said compoundis selected from the group consisting of 6-nitroso-1,2-benzopyrone,3-nitrosobenzamide, 5-nitroso-1(2H)-isoquinolinone,7-nitroso-1(2H)-isoquinolinone, and 8-nitroso-1(2H)-isoquinolinone. 17.A method according to claim 16, wherein said compound is3-nitrosobenzamide.
 18. A method according to claim 17 wherein saidvirus is a retrovirus.
 19. A method according to claim 18 wherein saidretrovirus is HIV.
 20. A method of reducing the level of integratedviral DNA from the genome of a host, said method comprisingadministering a pharmaceutically effective amount of a nitroso compoundwherein said compound is selected from the group consisting ofa compoundhaving the formula: ##STR16## wherein R₁, R₂, R₃, R₄, R₅ and R₆ areselected from the group consisting of hydrogen and nitroso, and only oneof R₁, R₂, R₃, R₄, R₅ and R₆ is a nitroso group, a compound having theformula: ##STR17## wherein R₁, R₂, and R₃ are selected from the groupconsisting of hydrogen and nitroso, and only one of R₁, R₂, and R₃ is anitroso group, and a a compound having the formula: ##STR18## whereinR₁, R₂, R₃, R₅, and R₆ are selected from the group consisting ofhydrogen and nitroso, and only one of R₁, R₂, R₃, R₄, and R₅ is anitroso group.
 21. A method according to claim 20, wherein said compoundis selected from the group consisting of 6-nitroso-1,2-benzopyrone,3-nitrosobenzamide, 5-nitroso-1(2H)-isoquinolinone,7-nitroso-1(2H)-isoquinolinone, and 8-nitroso-1(2H)-isoquinolinone. 22.A method according to claim 21, wherein said compound is3-nitrosobenzamide.
 23. A method according to claim 22 wherein saidvirus is a retrovirus.
 24. A method according to claim 23 wherein saidretrovirus is HIV.